Total chemical synthesis and NMR characterization of the glycopeptide tx5a, a heavily post‐translationally modified conotoxin, reveals that the glycan structure is α‐d‐Gal‐(1→3)‐α‐d‐GalNAc

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Abstract
The 13‐amino acid glycopeptide tx5a (Gla‐Cys‐Cys‐Gla‐Asp‐Gly‐Trp*‐Cys‐Cys‐Thr*‐Ala‐Ala‐Hyp‐OH, where Trp* = 6‐bromotryptophan and Thr* = Gal‐GalNAc‐threonine), isolated from Conus textile, causes hyperactivity and spasticity when injected intracerebral ventricularly into mice. It contains nine post‐translationally modified residues: four cysteine residues, two γ‐carboxyglutamic acid residues, and one residue each of 6‐bromotryptophan, 4‐trans‐hydroxyproline and glycosylated threonine. The chemical nature of each of these has been determined with the exception of the glycan linkage pattern on threonine and the stereochemistry of the 6‐bromotryptophan residue. Previous investigations have demonstrated that tx5a contains a disaccharide composed of N‐acetylgalactosamine (GalNAc) and galactose (Gal), but the interresidue linkage was not characterized. We hypothesized that tx5a contained the T‐antigen, β‐d‐Gal‐(1→3)‐α‐d‐GalNAc, one of the most common O‐linked glycan structures, identified previously in another Conus glycopeptide, contalukin‐G. We therefore utilized the peracetylated form of this glycan attached to Fmoc‐threonine in an attempted synthesis. While the result‐ing synthetic peptide (Gla‐Cys‐Cys‐Gla‐Asp‐Gly‐Trp*‐Cys‐Cys‐Thr*‐Ala‐Ala‐Hyp‐OH, where Trp* =6‐bromotryptophan and Thr* = β‐d‐Gal‐(1→3)‐α‐d‐GalNAc‐threonine) and the native peptide had almost identical mass spectra, a comparison of their RP‐HPLC chromatograms suggested that the two forms were not identical. Two‐dimensional 1H homonuclear and 13C‐1H heteronuclear NMR spectroscopy of native tx5a isolated from Conus textile was then used to determine that the glycan present on tx5a indeed is not the aforementioned T‐antigen, but rather α‐d‐Gal‐(1→3)‐α‐d‐GalNAc.